Characteristics of patients
The clinical characteristics of patients are summarized in Table 1. Patients with alcoholic cirrhosis (53.6 ± 15.62 years of age) and those with mixed cirrhosis (52.21 ± 6.30 years of age) tended to be elderly individuals, while binge drinkers were mainly among the younger age group (28.34 ± 9.59 years). Moreover, male patients were more likely to drink than female patients. Liver biochemistry analyses, including alanine aminotransferase (ALT), aspartate transaminase (AST), total bilirubin (TBIL), γ-glutamyl transferase (GGT), alkaline phosphatase (ALP), and albumin (ALB), showed alterations in patients with cirrhosis. Interestingly, the proportion of neutrophils in binge drinkers was significantly increased.
Circulating CD4+, and CD8+ MAIT cells are depleted in patients with alcoholic cirrhosis and mixed cirrhosis
Human MAIT cells were gated on CD3+CD161+Vα7.2+ lymphocytes (Figure 1A). The frequency of MAIT cells was significantly depleted in patients with alcoholic fatty liver diseases (p < 0.0001), alcoholic cirrhosis (p = 0.003) and mixed cirrhosis (p = 0.002), compared with HCs (Figure 1B). However, no obvious reductions in the frequency of MAIT cells were observed among binge drinkers (p = 1) and heavy drinkers without overt liver diseases (p = 0.982). The frequency of MAIT cells were comparable in patients with alcoholic cirrhosis and mixed cirrhosis (p = 0.903). Furthermore, we found a significant reduction in the frequency of MAIT cells in patients with chronic HBV infection compared with healthy controls (p = 0.029). No significant differences were observed in patients with chronic HBV infection, alcoholic cirrhosis, or mixed cirrhosis (Supplementary Figure 1). We also used the 5-OP-RU tetramer and TCRβ to confirm our gating strategy and found no significant differences between the two gating strategies (p = 0.065) (Figure 1C).
The MAIT cells are divided into subsets, based on CD4 and CD8 expression 20.Our further analysis indicated that the percentage of CD8+, CD4+, and CD4-CD8-(double negative, DN) MAIT cells were comparable among patients with alcoholic cirrhosis, mixed cirrhosis, and HCs (Figure 1D). In patients with cirrhosis, CD8+ and CD4+ MAIT cells among the T cells were both lower compared with HCs. In addition, no significant associations were noted between gender (p = 0.656) or age (p = 0.504) and MAIT cell frequency in HCs (Supplementary Figure 2). We also divided patients with alcoholic fatty liver disease and alcoholic cirrhosis into 2 groups based on alcohol cessation (Figure 1E). However, MAIT cells in patients with ALD who had abstained from alcohol showed no significant changes, compared with patients who had abstained from alcohol (p = 0.076). Thus, MAIT cells are depleted in chronic liver diseases.
Correlation between MAIT cell frequency and liver function in patients with cirrhosis
To assess the clinical significance of reduced MAIT cells in patients with alcoholic cirrhosis and mixed cirrhosis, we performed Spearman’s correlation analysis. Based on changes in clinical parameters presented in Table1, we gained further insights regarding AST, GGT, TBIL, ALP, and albumin levels. Notably, a negative correlation was found between MAIT cell frequency and AST (p = 0.02), GGT (p < 0.001), and TBIL (p = 0.004) levels in patients with alcoholic cirrhosis, while no correlation was noted between ALB levels (p = 0.747) (Figure 2A). In addition, a negative correlation was noted between MAIT cell frequency and AST and, TBIL among all patients with cirrhosis. The correlation between MAIT cell frequency and GGT was not as evident as that among patients with alcoholic cirrhosis (Figure 2B).
To assess the diagnostic ability of MAIT cell frequency as it relates to validated markers of liver function, we constructed a receiver operating characteristic curve. We evaluated the diagnostic value of MAIT cells for cirrhosis, and determined the area under the curve, sensitivity, specificity, and cut-off values to be 0.703, 49.4%, 83.1%, and 2.805%, respectively (Figure 2C). In order to discriminate between patients with cirrhosis and Child-Pugh A or Child-Pugh B or, C, we constructed a ROC characteristic, which yielded an area under the curve, sensitivity, specificity and cut-off values of 0.687, 65.2%, 70.8%, and 1.515%, respectively (Figure 2D). Taken together, our data reveal a negative correlation between circulating MAIT cell frequency and liver function, and support the notion that MAIT can be a biomarker to evaluate liver injury in ALD.
Activated MAIT cells display an altered cytokine profile and cytotoxic ability in patients with alcoholic cirrhosis
We investigated the phenotype and effector functions of circulating MAIT cells in HCs and patients with ALD. We determined that MAIT cells in the blood were activated in patients with cirrhosis by the elevation of CD69 levels (Figure 3A). As T cell activation is associated with a change in effector function, we analyzed the ability of MAIT cells to produce cytokines and cytolytic proteins after PMA-ionomycin stimulation. Compared with HCs, IL-17 production was elevated in MAIT cells of patients with alcoholic cirrhosis (p = 0.02). The MAIT cells of patients with alcoholic cirrhosis produced markedly lower levels of TNF-α (p = 0.033). Moreover, MAIT cells from patients with ALD showed higher perforin levels (p = 0.046). In contrast, IFN-γ and granzyme B production were comparable among MAIT cells in the blood samples of all subjects (Figure 3B, 3C). Taken together, MAIT cells in patients with alcoholic cirrhosis may be highly activated, which results in an altered cytokine profile and cytotoxic ability.
Elevated IL-8 levels combined with elevated IL-12 and IL-18 levels promote apoptosis of MAIT cells in vivo and in vitro
We tried to determine the underlying cause for the reduced circulating MAIT cell frequency in patients with ALD. Previous studies have demonstrated that MAIT cells may be recruited to the liver in chronic liver diseases, which can be identified by elevated levels of CCR6 and CXCR6 (20,21). However, we observed no increase in levels of CCR6 and CXCR6 on circulating MAIT cells in patients with alcoholic and mixed cirrhosis (Figure 4A). Furthermore, previous studies have shown that apoptosis (22) or exhaustion (19,23) may lead to the reduction in MAIT cell frequency. The expression of the immune checkpoint inhibitory molecule PD-1, often indicates T cell exhaustion (20,23). However, PD-1 expression on circulating MAIT cells showed no significant differences between patients with cirrhosis and HCs (Figure 4B). As shown in Figure 4C, MAIT cells were prone to apoptosis in patients with alcoholic cirrhosis (p = 0.034).
Cytokine-dependent pathways may contribute to MAIT loss (17,19). We further quantified cytokines and chemokines in HCs, and patients with alcoholic cirrhosis, and mixed cirrhosis, as well as binge drinkers (Table 2). As shown in Table 2, IL-12 and IL-23, which activate MAIT cells, were significantly increased in patients with cirrhosis. The concentration of IL-18, which is associated with the activation and exhaustion of MAIT cells (24,25), was considerably elevated in all groups. In patients with alcoholic cirrhosis, levels of the proinflammatory chemokine, IL-8, were significantly increased compared with those in HCs (Figure 4D). Moreover, MAIT cell frequency was inversely correlated with the levels of circulating IL-8 in patients with alcoholic cirrhosis (Figure 4E). The PBMCs of healthy subjects were exposed to increasing doses of IL-8 and subjected to 24 h of culture. We found that different levels of IL-8 did not promote apoptosis in MAIT cells (Supplementary Figure 3). Furthermore, we cultured PBMCs from HCs with IL-12, IL-18, and IL-8. Our results indicated that IL-12 and IL-18, combined with IL-8 can significantly enhance apoptosis of MAIT cells (Figure 4F). Collectively, these data suggest that the inflammatory environment in alcoholic cirrhosis could promote apoptosis in MAIT cells.